两种片形吸虫成虫抗原组分分析及其免疫学鉴定  

Analysis and immunological identification of antigenic components of Fasciola hepatica and Fasciola gigantica adults

作　　者：陈凤 艾琳 陈家旭 沈慧敏 赵银娇 刘榆华 陈绍荣 罗家军 罗天鹏 周晓农 CHEN Feng;AI Lin;CHEN Jia-xu;SHEN Hui-min;ZHAO Yin-jiao;LIU Yu-hua;CHEN Shao-rong;LUO Jia-jun;LUO Tian-peng;ZHOU Xiao-nong(Institute of Schistosomiasis Control, Dali Prefecture,Yunnan Province,Dali Yunnan,China 671000;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Chinese Center for Tropical Disease Research,Key Laboratory of Parasite and Vector Biology,Ministry of Health,WHO Collaborating Center of Tropical Disease,Shanghai,China 200025)

机构地区：[1]云南省大理州血吸虫病防治研究所,云南大理671000 [2]中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,卫生部寄生虫与病原生物学重点实验室,世界卫生组织热带病合作中心

出　　处：《中国病原生物学杂志》2018年第11期1211-1215,共5页Journal of Pathogen Biology

基　　金：卫生部寄生虫病原与媒介生物学重点实验室开放课题(No.WSBKTKT-201602)

摘　　要：目的应用SDS-PAGE对肝片形吸虫和巨片形吸虫成虫蛋白组分进行比较分析,并Western blot检测其特异性蛋白分子。方法分别收集肝片形吸虫和巨片形吸虫成虫,冰上研磨匀浆,提取上清蛋白,采用SDS-PAGE分析肝片形吸虫和巨片形吸虫差异蛋白组分;应用Western blot检测特异蛋白组分。结果虫体蛋白经SDS-PAGE后采用Gel Doc XR+凝胶成像系统对电泳图像进行高灵敏度分析,巨片形吸虫和肝片形吸虫成虫均有37条带,低灵敏分析显示肝片形吸虫成虫蛋白主要7条带,巨片形吸虫成虫蛋白主要有6条带,相对分子质量集中在10×10^3~70×10^3。Western blot检测巨片形吸虫有6条反应带,分别在150×10^3、100×10^3、75×10^3、50×10^3、34×10^3、23×10^3等位置;肝片形吸虫5条反应带,分别在55×10^3、37×10^3、34×10^3、23×10^3、15×10^3等位置。与血吸虫、囊虫、广州管圆线虫、旋毛虫的交叉反应蛋白为75×10^3、100×10^3、75×10^3组分(巨片形吸虫)和60.34×10^3组分(肝片形吸虫)。结论肝片形吸虫成虫与巨片形吸虫成蛋白组成有差异不明显,抗原特异的蛋白在23×10^3、15×10^3等位置,且22×10^3~24×10^3组分占比较大(在在肝片形吸虫占48.4%,巨片形吸虫占77.3%)。其特异的蛋白酶类有待经质谱分析后用于诊断抗原及疫苗研究,而15×10^3组分可能是肝片形吸虫特有的可区别于巨片形吸虫的蛋白。Objective To use SDS-PAGE to compare the protein components of Fasciola hepatica and F.gigantica in order to identify specific molecular bands using immunoblotting.Methods Adults of F.hepatica and F.gigantica were collected separately and homogenized on ice to obtain a homogenate.The supernatant was assayed for protein contents and analyzed using SDS-PAGE to ascertain the difference in the molecular mass of components of both trematodes and their macromolecular protein bands.Moreover,specific protein bands were selected using Western blotting.Positive bands were selected for comparison to protein bands on SDS-PAGE gels.Results After different proteins were separated using SDS-PAGE,37 bands produced by both F.hepatica and F.gigantica were analyzed with a high level of sensitivity using the Gel Doe XR+ gel imaging system.Seven bands produced by F.hepatica and 6 produced by F.gigantica with a relative molecular mass of 10-70kDa were analyzed with a low level of sensitivity.F.gigantica produced 6 main bands with a molecular mass of approximately 150,100,75,50,34,and 23 kDa,and F.hepatica produced 5 main bands with a molecular mass around 55,37,34,23,and 15kDa.F.gigantica components with a molecular mass of 75, 100,and 75kDa and an F.hepatica component with a molecular mass 60.34kDa cross-reacted with proteins from Schistosoma,Cysticercus,Angiostrongylus cantonensis,and Trichinella spiralis.Conclusion The protein components of adult F.hepatica and F.gigantica did not differ significantly.Antigens produced specific protein bands at 23kDa and 15 kDa;a large proportion of these bands was at 22-24kDa (48.4% for F.hepatica and 77.3% for F.gigantica).Specific proteases need to be used to identify antigens and vaccines using mass spectrometry.However,a protein band at 15 kDa may be produced by a protein specific to F.hepatica that can be used to distinguish it from F.gigantica.

关 键 词：片形吸虫 抗原 SDS-PAGE WESTERN BLOT 

分 类 号：R383.2[医药卫生—医学寄生虫学]